Mammary glands undergo extensive proliferation, branching and terminal differentiation during pregnancy to prepare for lactation, and there is growing evidence that stem cells at least partly drive this process. There is also growing interest in the connection between stem cells and cancer. The apparent similarity between the ability of cancer cells to grow uncontrollably and that of the stem cells to continuously self-renew has led to the notion that cancers are diseases of stem cells. It posits that only a fraction of the cells within the tumor mass can generate tumors and such cancer stem cells cause relapse and prevent cure. Indeed, stem-cell markers are expressed by a small but highly tumorigenic subset of breast carcinoma cells. In this application, we seek to perform exploratory experiments - using mammalian numb proteins and the insights we obtained from our studies in the nervous system as entry point - to examine the contribution of two modes of cell division - symmetric vs. asymmetric - in regulating the behavior of putative stem cells during mammary gland growth, as a means to identify markers and essential regulators of mammary stem cells. If successful, our research will not only help us to better understand mammary development but also point to new avenues for exploring the connection between stem cells and tumor cells and provide insights for devising novel therapeutic measures for treating breast cancer. Asymmetric cell division is an attractive means for stem cells to balance self-renewal and differentiation - namely, by producing one daughter cell that remains as a stem cell and the other that differentiates. In invertebrates like Drosophila, the Numb protein makes the two daughter cells different after an asymmetric division by segregating primarily into one daughter cell to promote its fate. Recent findings from our laboratory show that neural stem (progenitor) cells use asymmetric numb segregation to balance self-renewal and differentiation during mouse embryogenesis. Mammalian numb proteins are encoded by two genes, m-numb (Numb) and numblike (Numbl), which have largely overlapping functions. When both Numb and Numbl are mutated, neural progenitor cells lose their ability to self-renew as both daughter cells become neurons. Conversely, forcing numb proteins to segregate symmetrically into both daughter cells inhibits neuron production by forcing both to choose self-renewal. In this exploratory R21 application, we hypothesize that numb-mediated asymmetric cell divisions are also used by stem/progenitor cells when they drive mammary gland development during pregnancy and that markers and essential regulators of mammary stem cells can be identified using mutant mammary glands in which the ratio between stem/progenitor and differentiated cells is skewed by changes in numb segregation. Aim 1 will examine the importance of numb-mediated asymmetric cell division in mammary gland development by using Cre-loxP-mediated gene targeting to replace the endogenous numb proteins with individual variants that segregate either symmetrically or asymmetrically. Aim 2 intends to identify markers and essential regulators of mammary stem cells by comparing gene expression profiles between mammary glands in which the two numb genes are mutated and those containing only a symmetrically segregating numb protein. PUBLIC HEALTH RELEVANCE: A fundamental issue in cancer biology and treatment is why killing the vast majority of cancer cells do not lead to a cure. The apparent similarity between tumor cells and stem cells has led to the notion that cancers are diseases of stem cells. It is believed that only a fraction of the cells within the tumor mass is capable of generating tumors. Since stem cells have limitless capability to self-renew and generate large numbers of progeny, a few remaining cancer stem cells after treatment may be sufficient to form new tumors and, consequently, cause relapse and prevent cure. In this application, we seek to perform exploratory experiments to identify genes and cellular mechanisms that regulate the behavior of stem cells during mammary gland growth. If successful, our research will not only help us to better understand mammary development but also point to new avenues for exploring the connection between stem cells and tumor cells and provide insights for devising novel therapeutic measures for treating breast cancer.